Toxoplasma gondii rhomboid protein 1 (TgROM1) is a potential vaccine candidate against toxoplasmosis

Veterinary Parasitology 184 (2012) 154–160

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Toxoplasma gondii rhomboid protein 1 (TgROM1) is a potential vaccine candidate against toxoplasmosis Jianhua Li 1 , Qianzhong Han 1 , Pengtao Gong, Tuo Yang, Baoyan Ren, Shijie Li, Xichen Zhang ∗ College of Animal Science and Veterinary Medicine, Jilin University, 130062 Changchun, Jilin, China

a r t i c l e

i n f o

Article history: Received 11 May 2011 Received in revised form 4 August 2011 Accepted 9 August 2011 Keywords: Toxoplasma gondii DNA vaccine Rhomboid BALB/c mice

a b s t r a c t Infection with the intracellular protozoan parasite Toxoplasma gondii causes serious public health problems and is of great economic importance worldwide. The rhomboid proteins which are responsible for adhesion and invasion of host cells have been suggested as vaccine candidates against toxoplasmosis. A DNA vaccine (pVAX-ROM1) encoding T. gondii rhomboid protein 1 (TgROM1) gene was constructed and the immune response and protective efficacy of this vaccine against lethal challenge in BALB/c mice were evaluated. The results indicated that specific antibody and lymphocyte proliferative responses were elicited in mice receiving pVAX-ROM1. The production levels of IFN-␥, IL-2, IL-4, and IL-10, as well as the percentage of CD4+ cells in mice vaccinated with pVAX-ROM1 were significantly increased respectively, compared to controls receiving either pVAX1 alone or PBS. After lethal challenge, the mice immunized with pVAX-ROM1 showed an increased survival time compared with the mice in the controls. Our data suggested that a DNA vaccine pVAX-ROM1 encoding T. gondii rhomboid protein 1 triggered strong humoral and cellular responses, and prolonged survival time against T. gondii infection in BALB/c mice. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Toxoplasma gondii is the agent of toxoplasmosis that has a major significance from veterinary and public health perspectives. In pregnant women, T. gondii infection can lead to miscarriages, neonatal malformations, ocular complications and severe cognitive impairments in the fetus (Cook, 1990; Dodds, 2006). Furthermore, T. gondii infection may cause serious clinical manifestations in AIDS patients (Yuan et al., 2007; Elsheikha, 2008). Animals during pregnancy infected with T. gondii, especially in sheep, often result in abortion, causing considerable economic losses. These infected animals, mainly felines and pigs, also represent an

∗ Corresponding author. Fax: +86 431 87981351. E-mail addresses: [email protected] (J. Li), [email protected] (X. Zhang). 1 These authors contributed equally to this study. 0304-4017/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2011.08.014

important source of transmission to humans (Tenter et al., 2000; Dubey et al., 2005; Dubey and Jones, 2008; Zou et al., 2009). Treatment of toxoplasmosis is difficult due to the toxicities of available drugs. Under the present scenario, development of an effective vaccine was an attractive alternative (Bhopale, 2003). There is no vaccine available against human infection with T. gondii, only a live attenuated vaccine has been available for veterinary use in sheep for several years in some countries. However, this live attenuated vaccine is expensive and has the possibility to revert to a pathogenic form (Mateus-Pinilla et al., 1999; Liu et al., 2008). In order to overcome these problems, DNA vaccines were proposed recently (Bout et al., 2002). Several studies have demonstrated that DNA vaccination with genes encoding T. gondii antigens showed certain protection and increased the survival time of mice and reduced the number of brain cysts in rodents (Vercammen et al., 2000; Leyva et al., 2001; Dautu et al., 2007; Jongert, 2007; Zhang

J. Li et al. / Veterinary Parasitology 184 (2012) 154–160

et al., 2007; Xue et al., 2008; Fang et al., 2009; Liu et al., 2009; Wang et al., 2009). Despite intensive efforts, development of an effective vaccine against this protozoan parasite remains a long-term goal. Apicomplexan pathogens replicate exclusively within the confines of a host cell. Invasion into the host cells requires an array of specialized parasite molecules, many of which have long been considered to have potential as targets of drug or vaccine-based therapies (O’Donnell and Blackman, 2005; Dowse et al., 2008). Among the targets that have been studied, rhomboid proteases seem promising (Kim, 2004; Freeman, 2008). Rhomboids are a recently discovered family of widely distributed intramembrane serine proteases that have diverse biological functions, including the regulation of growth factor signaling, mitochondrial fusion, and parasite invasion (Freeman, 2008). Recent studies have suggested that proteases played critical roles in the process of apicomplexan parasites invasion (Brossier et al., 2005; Carruthers and Blackman, 2005; Dowse et al., 2008). Rhomboid proteases were found in Toxoplasma and Plasmodium to cleave cell adhesins that were essential for invasion (Urban and Freeman, 2003; Kim, 2004; Zhou et al., 2004; Dowse et al., 2008; Baker et al., 2006). T. gondii contains six rhomboids that are expressed in different life cycle stages and localized to different cellular compartments (Brossier et al., 2005). Toxoplasma rhomboid protein 1 (TgROM1) was localized in the secretory pathway of the parasite, including the Golgi apparatus and micronemes, which contained adhesive proteins involved in invasion of host cells. It played an important role in parasite growth in vitro by the tetracycline-repressible system and required for efficient intracellular growth by T. gondii (Brossier et al., 2008). Although rhomboid proteins are related to T. gondii invasion process, protective efficacy as vaccine antigens against parasitic diseases remains unclear. In the present study, we constructed a eukaryotic plasmid pVAX-ROM1 encoding T. gondii rhomboid protein 1. The immune responses in BALB/C mice and its protective efficacy against lethal challenge with the highly virulent RH strain of T. gondii were examined. 2. Materials and methods 2.1. Parasites and polyclonal antibody T. gondii tachyzoites (highly virulent RH strain) were propagated by intraperitoneal passages through 6 weeks old specific-pathogen-free (SPF) female BALB/c mice. All experimental procedures were conducted according to the appropriate guidelines of the Center of Experimental Animals, Jilin University. Tachyzoites were harvested from the peritoneal fluid of the mice at day 3 after infected with 1 × 103 tachyzoites. For preparing toxoplasma tachyzoites lysate antigen (TLA), tachyzoites were disrupted by sonication on ice (400 W × 5 min) and centrifuged at 2100 × g for 10 min at 4 ◦ C. The supernatant was collected as TLA for polyclonal antibody, ELISA and lymphocyte proliferative response analysis. Anti-T. gondii tachyzoites polyclonal antibody was prepared in a standard procedure. Briefly, each BALB/C mouse was first injected with 100 ␮g of TLA

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emulsified with an equal volume of Freund’s complete adjuvant. Each mouse was injected again at the second week with the same amount of antigen mixed with an equal volume of Freund’s incomplete adjuvant. Two weeks later, a boost injection from immunized mice and one week later polyclonal antibody was collected. 2.2. pVAX-ROM1 plasmid construction The complete TgROM1 open reading frame (ORF) (GenBankTM accession No. AY587210) was obtained by PCR amplification with genomic DNA as template, using primers: (forward primer): 5 GCTTCGAACCGATTCAACGAGCAGT-3 , (reverse primer): 5 -GCGAATTCGGTAAGTAATCAGGGAGGG-3 , in which the HindIII and ECoR I restriction sites were introduced, respectively. The amplified DNA fragment was inserted into pMD18-T vector (TaKaRa, China) to form a recombinant plasmid pMD-ROM1. The TgROM1 ORF fragment was subcloned into eukaryotic expression vector pVAX1 to form plasmid pVAX-ROM1 through HindIII and ECoR I sites. 2.3. pVAX-ROM1 plasmid expression in vitro Recombinant plasmid pVAX-ROM1 was transfected into Hela cells with lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions. Forty-eight hours post transfection, cells were fixed with 100% acetone for 30 min and washed with PBS-0.1% Triton-X-100 (PBST) for three times, then blocked in 10% goat serum for 2 h. The cells were incubated with mouse anti-T. gondii tachyzoites polyclonal antibody at 37 ◦ C for 2 h. After washing, cells were incubated with fluorescein isothiocyanate (FITC) labeled goat anti-mouse IgG antibody (1:2000) (Boster Biological Technology, Wuhan, China) at 37 ◦ C for 1 h. Fluorescence was examined under a fluorescence microscope (Carl Zeiss, Germany). Hela cells transfected with pVAX1 served as the negative control. 2.4. DNA vaccination Four weeks old female BALB/c mice were randomly divided into three groups (25 mice in each group). Mice in different groups were intramuscularly injected with pVAX-ROM1 (100 ␮g/each), the empty vector pVAX1 (100 ␮g/each), and PBS (100 ␮l/each), respectively. All groups were vaccinated three times at weeks 0, 2 and 4, respectively. 2.5. Specific antibodies response against T. gondii Serum samples from 10 randomly chosen mice in each group were collected from venous plexus of mice tails at weeks 0, 2, 4 and 6 for ELISA. The microtiter plates were coated overnight at 4 ◦ C with crude T. gondii tachyzoites antigens, blocked with 5% bovine serum albumin (BSA) in PBS at room temperature for 2 h, and incubated with sera diluted 1:160 in 1% BSA-PBS at 37 ◦ C for 1 h. The plates were washed with PBST and incubated with HRPlabeled goat anti-mouse IgG antibody (1:2000) at 37 ◦ C

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for 1 h. After washing substrate solution containing H2 O2 , citrate-phosphate and O-phenylenediamine (OPD) were added and the reaction was stopped by 2 M H2 SO4 . The absorbance at 450 nm was measured using a microplate ELISA reader. All samples were run in triplicate. 2.6. Splenocyte proliferation responses Splenocyte suspension was obtained from 5 mice in each group at week 6 by gentle squeezing of the whole spleens in PBS buffer. The cells (5 × 105 cells/well) were cultured in DMEM medium supplemented with 10% FCS and stimulated with TLA (10 ␮g/ml) at 37 ◦ C for 48 h at 5% CO2 . After incubated with Alamar blue (10 ␮l/well) for 12 h, the plates were read at 570 nm with an ELISA reader. Stimulation index (SI) was calculated by the ratio of OD570 nm of stimulated cells/OD570 nm of unstimulated cells. 2.7. Productions of cytokines The productions of cytokines from splenocytes stimulated by TLA at different time were detected by ELISA according to the manufacturer’s instructions (Jiamay Biotech, China). The supernatants from the reactions were collected by centrifugation and analyzed for the activities of interleukin-2 (IL-2) and IL-4 at 24 h, IL-10 at 72 h, and gamma-interferon (IFN-␥) at 96 h following described previously procedures (Fachado et al., 2003; Mévélec et al., 2005; Dautu et al., 2007; Cong et al., 2008; Cuppari et al., 2008; Xue et al., 2008; Liu et al., 2010a,b). 2.8. Percentages of CD4+ and CD8+ T lymphocytes Two weeks after the last immunization, suspensions of splenocytes were obtained by gentle squeezing from spleens of 5 mice in each group in FACS buffer (phosphatebuffered saline with 5% fetal calf serum, and 0.1% sodium azide). Residual debris was removed by centrifugation. Cells suspension was stained with optimal concentrations of FITC conjugated anti-immunoglobulin (Ig) antibody (100 ␮g/mL), PE conjugated anti-CD4 antibody (5 ␮g/mL), PE conjugated anti-CD8 antibody (12.5 ␮g/mL), respectively (PharMingen, San Diego, CA). After counterstained with propidium iodide (PI), cells were analyzed by flow cytometry (FACSan; Becton-Dickinson Immunocytometry Systems, Mountain View, CA). 2.9. Protective efficacy of DNA vaccine against T. gondii Two weeks after the last immunization, 10 mice were selected randomly from each group and each mouse was challenged with a lethal dose of 1 × 103 tachyzoites of T. gondii (RH strain) by intra peritoneal injections. The survival time for each mouse was recorded and the percentages of mice survived were calculated. 2.10. Statistical analysis Statistical analysis was performed by variance (ANOVA) and Duncan’s multiple range tests using SPSS 14.0 software.

Fig. 1. Construction of plasmid pVAX-ROM1. TgROM1-DNA fragment was amplified by PCR and inserted into pVAX1 at the sites of HindIII and ECoR I.

Differences were considered to be statistically significant between two groups when P < 0.05. 3. Results 3.1. Construction of recombinant plasmid pVAX-ROM1 The complete TgROM1 ORF fragment was amplified by PCR and inserted into pVAX1 eukaryotic expression vector and the resultant plasmid was named pVAX-ROM1 (Fig. 1). The insert of TgROM1 ORF was verified by DNA sequencing. 3.2. TgROM1 protein expression in Hela cells detected by IFA After immune-fluorescence staining, green fluorescence could be seen in Hela cells transfected with plasmid pVAX-ROM1 48 h post transfection, but not in cells transfected with pVAX1 vector plasmid. The results indicated that recombinant TgROM1 protein has been expressed successfully in Hela cells (Fig. 2). 3.3. Evaluation of humoral immunity Two weeks after the third immunization, the OD values of specific IgG reached to a very high level in those mice immunized with pVAX-ROM1 (Fig. 3). Compared to pVAX1or PBS controls, the differences of OD values in pVAX-ROM1 were significant (P < 0.05). The results showed that vaccination with plasmid pVAX-ROM1 induced a specific antibody response.

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Fig. 2. Indirect immunofluorescence detection of recombinant TgROM1 protein expression on Hela cells. Hela cells were transfected with pVAX-ROM1 (A) and empty vector pVAX1 (B), respectively.

3.4. Evaluation of splenocyte proliferation Proliferation SI from mice immunized with pVAX-ROM1 was estimated to be 1.22 ± 0.05, which was significantly higher than that with PBS (0.41 ± 0.06) or pVAX1 (0.49 ± 0.08) (P < 0.05; N = 5).

3.5. Evaluation of cytokines productions The average expression levels of IFN-␥, IL-2, IL-4, and IL-10 were estimated to be 454.56 pg/ml, 383.45 pg/ml, 239.64 pg/ml, and 422.89 pg/ml from pVAX-ROM1 group, which were significantly higher than that from PBS and pVAX1groups (P < 0.05; N = 5), as shown in Fig. 4.

Fig. 4. Cytokines production from splenocytes after TLA stimulation in vitro. Splenocyte was obtained at day 14 after the final immunization. Data are expressed as mean ± S.E. (n = 5). Statistically significant differences (P < 0.05) are indicated by (**).

3.6. Evaluation of the percentages of CD4+ and CD8+ cells The percentages of CD4+ and CD8+ lymphocytes (CD4+ with 35.98 ± 0.66; CD8+ with 14.42 ± 0.62 in pVAX-ROM1 group were higher than those in PBS group (CD4 + with 19.10 ± 0.72; CD8+ with 11.96 ± 0.71) or in pVAX1 group (CD4+ with 22.32 ± 0.82; CD8+ with 12.34 ± 0.69). There was a significant difference in terms of the percentages of CD4+ lymphocytes in pVAX-ROM1 group when compared to PBS or pVAX1 groups (P < 0.05), but no significant difference in the percentages of CD8+ lymphocytes among different groups. The data was shown in Table 1. Table 1 The percentages of CD4+ and CD8+ T cells from immunized BABL/c mice. Fig. 3. Humoral immune responses induced by pVAX-ROM1 DNA vaccination. BALB/c mice were immunized with pVAX-ROM1, pVAX1 vector, and PBS under the same conditions. Serum samples from 10 randomly chosen mice from each group were detected for specific anti-T. gondii tachyzoites antibodies with ELISA assays. OD readings of sera at A450 diluted at 1:160 were shown in the figure. Each bar represents the mean OD (±S.E., n = 10). Statistically significant differences of OD values (P < 0.05) are indicated by (**).

Groups

CD4+ (%: mean ± S.E., n = 5)

CD8+ (%: mean ± S.E., n = 5)

pVAX-ROM1 (A) pVAX1 (B) PBS (C)

35.98 ± 0.66** 22.32 ± 0.82 19.10 ± 0.72

14.42 ± 0.62 12.34 ± 0.69 11.96 ± 0.71

**

C.

Note: The percentage of CD4+ : P < 0.05 as compared with groups B and

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120

PBS pVAX1

% Survival

100

pVAX-ROM1

80 60 40 20 0 1

3

5

7

9

11

13

15

Days after challenge Fig. 5. Survival times of the immunized BALB/c mice after T. gondii tachyzoites challenge. Survival times were monitored daily after the challenge. The mice immunized with pVAX-ROM1 were dead from day 10 to 17. Negative controls mice immunized with pVAX1and PBS died within 5 days. All experimental BALB/c mice at day 17 were dead after infected with T. gondii tachyzoites. Mice immunized with the pVAX-ROM1 showed an increased survival time (12.5 ± 0.7 days) compared with mice in the control groups died within 5 days after challenge (P < 0.05).

3.7. Protective efficacy of DNA vaccination against T. gondii Mice immunized with pVAX-ROM1 showed an increased survival time (12.5 ± 0.7 days) compared to mice in controls receiving either PBS or pVAX1 died within 5 days after challenge (P < 0.05) (Fig. 5). The results indicated that immunization with pVAX-ROM1 could not effectively protect those mice from lethal T. gondii tachyzoites infection, only prolonged survival time in BALB/c mice. 4. Discussion In order to evaluate the role of TgROM1 against T. gondii infection, the recombinant plasmid pVAX-ROM1 was constructed and successfully expressed in vitro in Hela cells. Plasmid pVAX-ROM1 was able to elicit specific humoral and cellular immune responses. Though this DNA vaccine could not effectively protect BALB/c mice from lethal challenge it did prolonged the survival times of those immunized mice. In recent years, significant progress has been made in the identification of vaccine candidates in T. gondii which could elicit a protective immune response. These antigens included the dense granule proteins (GRA1, GRA2, GRA4, GRA6, GRA7, GRA8) (Desolme et al., 2000; Mévélec et al., 2005), rhoptry antigens (ROP1, ROP2) (Chen et al., 2003; Wang et al., 2007), matrix protein (MAG1) (Di Cristina et al., 2004), microneme proteins (MIC1, MIC3, MIC4, MIC6, MIC8) (Fang et al., 2009; Peng et al., 2009; Liu et al., 2010a,b), and surface antigens (SAG1, SAG2) (Mévélec et al., 2005; Lau and Fong, 2008). Rhomboid proteins play an important role in parasite invasion by their involvement in the proteolytic process and a potential role in the cleavage of microneme protein during host cell invasion (Brossier et al., 2005; Dowse and Soldati, 2005; Urban, 2006). TgROM1 was one of the rhomboid proteins expressed in the tachyzoite stage responsible for the disease (Brossier et al., 2005). TgROM1 was localized in the

secretory pathway of the parasite, including the Golgi apparatus and micronemes, which contains adhesive proteins involved in invasion of host cells. The location of TgROM1 within micronemes seems to preclude its role in processing of microneme proteins TMDs (Brossier et al., 2008). As an important molecule associated with the invasion process, no detailed studies have been reported as whether TgROM1 can be used as a DNA vaccine to induce immune responses and to provide protections against homologous challenge in mice. DNA vaccines against toxoplasmosis had been shown to be a powerful method for the induction of specific humoral immune responses (Angus et al., 2000; Vercammen et al., 2000; Leyva et al., 2001; Couper et al., 2003; Fachado et al., 2003; Dautu et al., 2007; Fang et al., 2009; Wang et al., 2009). Similar to those reports, specific IgG antibody responses against T. gondii were elicited in those mice receiving pVAX-ROM1 and the OD values of specific IgG reached to a high level at two weeks after the third immunization. As T. gondii is an obligate intracellular parasite, cellmediated immune responses involving both the innate and adaptive immune responses such as CD4+ and CD8+ T-cells and regulatory cytokines are known to be important in protecting the host and limiting multiplication of the parasite (Denkers and Gazzinelli, 1998). In this report, lymphocyte proliferative responses were elicited and the production levels of IFN-␥, IL-2, IL-4 and IL-10, as well as the percentage of CD4+ cells in mice receiving pVAX-ROM1 were significantly increased. The results indicated DNA pVAXROM1 could stimulate cell-mediated immune responses in BALB/c mice. CD 4+ lymphocytes as helper cells played a major role in the induction of protection (Gazzinelli et al., 1991). Results from this study also indicated that the percentage of CD4+ lymphocytes was increased. Furthermore, the development of Th1 lymphocytes is essential for cell-mediated immunity and resistance against intracellular pathogens. Both cytokines IL-2 and IFN-␥ primarily secreted through Th1 type cells. The productions of IL-2 and IFN-␥ reached high levels in pVAX-ROM1 group, suggesting a Th1 type immune response. However, if the secretion of these kinds of cytokines left unregulated, the same response can cause serious damage to host tissues and lead to mortality (Jankovic et al., 2010). IL-10 is a cytokine with broad antiinflammatory properties, one of which is to counteract the function of Th1 lymphocytes (Jankovic et al., 2010). Th1 cells also controlled themselves by producing IL-10 during T. gondii infection (O’Garra and Vieira, 2007). In the present study, the production of IL-10 also had a significant increase in pVAX-ROM1 vaccinated mice. The findings support the general concept that production of IL-10 is an important self-regulatory function of CD4+ T lymphocytes. As a factor of Th2 type immune response, a slight increase of cytokine IL-4 was also observed. Therefore, from the results, cellular immune responses triggered by DNA vaccine pVAX-ROM1 primarily skewed toward a Th1 type immune response. DNA vaccines encoding different T. gondii genes showed certain protection and extended the survival time of mice in several studies (Vercammen et al., 2000; Leyva et al., 2001; Dautu et al., 2007; Jongert, 2007; Zhang et al., 2007;

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Xue et al., 2008; Fang et al., 2009; Liu et al., 2009; Wang et al., 2009). Till now, there was no effective vaccine that produced complete protection against intra peritoneal challenge with the T. gondii RH strain (Angus et al., 2000; Leyva et al., 2001; Fachado et al., 2003). In this current study, pVAX-ROM1 could extend the survival time in BALB/c mice challenged with a lethal dose of T. gondii tachyzoites of virulent RH strain when compared to controls. The results from this study were similar in terms of protection efficacy to the results obtained from several other single gene DNA vaccines in BALB/c mice (Leyva et al., 2001; Fachado et al., 2003; Fang et al., 2009; Wang et al., 2009). In summary, our data suggested that DNA vaccine pVAX-ROM1 encoding T. gondii rhomboid protein 1 triggered strong humoral and cellular immune responses, prolonged survival time against T. gondii infection in BALB/c mice. Rhomboid protein1 warrant further studies as a potential vaccine candidate against T. gondii. Acknowledgments This work was supported by National Key Technology R & D Program of China (No. 2008BAD96B11-3) and Jilin Provincial Department of Science and Technology (No. 20100222). References Angus, C.W., Klivington-Evans, D., Dubey, J.P., Kovacs, J.A., 2000. Immunization with a DNA plasmid encoding the SAG1 (SAG1) protein of Toxoplasma gondii is immunogenic and protective in rodents. J. Infect. Dis. 181, 317–324. Baker, R.P., Wijetilaka, R., Urban, S., 2006. Two Plasmodium rhomboid proteases preferentially cleave different adhesions implicated in all invasive stages of malaria. PLoS Pathogens 2, e113. Bhopale, G.M., 2003. Development of a vaccine for toxoplasmosis: current status. Microbes Infect. 5, 457–462. Bout, D.T., Mevelec, M.N., Velge-Roussel, F., Dimier-Poisson, I., Lebrun, M., 2002. Prospects for a human Toxoplasma vaccine. Curr. Drug Targets Immune. Endocr. Metabol. Disord. 2, 227–234. Brossier, F., Jewett, T.J., Sibley, L.D., Urban, S., 2005. A spatially localized rhomboid protease cleaves cell surface adhesins essential for invasion by Toxoplasma. Proc. Natl. Acad. Sci. U.S.A. 102, 4146–4151. Brossier, F., Starnes, G.L., Wandy, L.B., Sibley, L.D., 2008. Microneme rhomboid protease TgROM1 is required for efficient intracellular growth of Toxoplasma gondii. Eukaryotic. Cell 7, 664–674. Carruthers, V.B., Blackman, M.J., 2005. A new release on life: emerging concepts in proteolysis and parasite invasion. Mol. Microbiol. 55, 1617–1630. Chen, H., Chen, G., Zheng, H., Guo, H., 2003. Induction of immune responses in mice by vaccination with liposome-entrapped DNA complexes encoding Toxoplasma gondii SAG1 and ROP1 genes. Chin. Med. J. (Engl.) 116, 1561–1566. Cong, H., Gu, Q.M., Yin, H.E., Wang, J.W., Zhao, Q.L., Zhou, H.Y., Li, Y., Zhang, J.Q., 2008. Multi-epitope DNA vaccine linked to the A2/B subunit of cholera toxin protect mice against Toxoplasma gondii. Vaccine 26, 3913–3921. Cook, G.C., 1990. Toxoplasma gondii infection: a potential danger to the unborn fetus and AIDS sufferer. Q. J. Med. 74, 3–19. Couper, K.N., Nielsen, H.V., Petersen, E., Roberts, F., Roberts, C.W., Alexander, J., 2003. DNA vaccination with the immunodominant tachyzoite surface antigen (SAG-1) protects against adult acquired Toxoplasma gondii infection but does not prevent maternofoetal transmission. Vaccine 21, 2813–2820. Cuppari, A.F., Sanchez, V., Ledesma, B., Frank, F.M., Goldman, A., Angel, S.O., Martin, V., 2008. Toxoplasma gondii protease inhibitor-1 (TgPI1) is a novel vaccine candidate against toxoplasmosis. Vaccine 26, 5040–5045. Dautu, G., Munyaka, B., Carmen, G., Zhang, G., Omata, Y., Xuenan, X., Igarashi, M., 2007. Toxoplasma gondii: DNA vaccination with genes

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